CN105194678A - Thermosensitive silica body and preparing method and application thereof - Google Patents
Thermosensitive silica body and preparing method and application thereof Download PDFInfo
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- CN105194678A CN105194678A CN201410281436.2A CN201410281436A CN105194678A CN 105194678 A CN105194678 A CN 105194678A CN 201410281436 A CN201410281436 A CN 201410281436A CN 105194678 A CN105194678 A CN 105194678A
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Abstract
The invention relates to a thermosensitive silica body and a preparing method and application thereof. The thermosensitive silica body and high-intensity focused ultrasound beams (HIFU) are combined and used for controlling release of hydrophobic drugs. The thermosensitive silica body is prepared from, by weight, 41.90-62.90 parts of lipid formed by the silica body, 40.00-60.00 parts of dipalmitoyl phosphatidyl choline, 5.20-8.50 parts of palmitoyl phosphatidyl choline and 14.00-19.50 parts of polyethylene glycol 2000-dipalmitoyl phosphatidyl ethanolamine. The thermosensitive silica body is high in stability and biocompatibility and capable of fast releasing the hydrophobic antineoplastic drugs when combined with HIFU and shows a remarkable therapeutic effect.
Description
Technical field
The invention belongs to drug controlled release field, specifically, the present invention relates to a kind of temperature-sensitive silica nodule and its production and use.
Background technology
At present, the whole world about 40% marketed drug and more than 80% research and development in new drug belong to poorly water soluble drugs.Due to poorly water-soluble, usually be difficult to the bioavailability reaching treatment requirement, or be difficult to make the preparation for intravenously administrable, medicine important is in a large number made not give full play to curative effect, cause that drug dose is large, curative effect is little, and serious toxic and side effects, greatly limit the application of poorly water soluble drugs.
And the existence of obstacle makes uniformly and effectively drug conveying to be become very complicated to knub position in tumor microenvironment, the drug dose of effectively killing needed for tumor cell at knub position clinically usually can cause Normocellular major injury and various side effect.Thermotherapy has shown it and has had enhancing pharmaceutical carrier exosmosing in solid tumor, can overcome the obstacle of drug delivery.The use of heat-sensitive carrier provides a kind of mechanism triggering high concentration active medicine and discharge rapidly in the target area.Thermal sensitive liposome (TSL), combines with an external heat source, such as microwave or infrared laser, and their contained medicines of region release that can raise at local tissue temperature, to improve sending of topical remedy.Thermal sensitive liposome and high temperature bond have presented good therapeutic effect.With can keep stable in Physiological temperatures range, but the non-temperature sensitive liposome (NTSL) not discharging medicine is compared, and thermal sensitive liposome can experience a phase transformation when heating, and presents higher permeability, discharges contained medicine.
High intensity focused ultrasonic beam (HIFU) can at the focal spot becoming a grade area apart from the position correction of wave source certain distance, for Noninvasive ablated tumor, knub position is carried out to the prolonged exposure of long period, produce heating ablation, thus direct killing tumor.Compared with other method, HIFU to fix a point stimulation tool as one, has unique advantage, because it can propagate into deep tissue, also can focus on specific position.This topical therapeutic depends on the stability of pharmaceutical carrier and the drug release ability of heat triggering, and to the accurate control etc. that temperature local in target area raises.Generally, be loaded with the carrier of medicine before its physical integrity is interfered, it can keep stable in physiological conditions.A desirable drug delivery process should be as follows: first, carrier load medicine circulates in blood; Then, the medicine that carries of bag under a suitable stimulation with certain speed by Co ntrolled release.
At present, high intensity focused ultrasonic beam (HIFU) is used to the drug release accelerating thermal sensitive liposome.Traditional thermal liposome causes drug release within the scope of about 42 DEG C-45 DEG C, and the time of drug release is greater than 30 minutes, and low temperature thermal sensitive liposome (LTSL) can discharge the medicine that its bag carries within the several seconds the temperature range of about 39 DEG C-40 DEG C.Because high intensity focused ultrasonic beam excites, larger liposome (>100nm) break and the generation of the comparatively fenestra shape defect of small liposome (<100nm) may be the main cause causing drug release; Also inertial cavitation effect can be there is between the light period.But the main shortcoming of liposome is deficient in stability in physiological conditions, and this often causes the medicine encapsulated to discharge too early before arrival target area.Therefore, the thermal sensitive liposome needing research and development more stable.
In recent years, silica nodule, a kind of novel organic-inorganic nano hybrid causes extensive concern.This biomimetic material derives from trialkoxy silane heterozygosis lipid, and it can form the bilayer vesicle structure closed as liposomes, is especially covalently covered with the atomic layer of siloxane backbone at silica nodule vesicle surface.Research shows: silica nodule can present the performance of good Drug controlled release, has high stability, acid and alkali-resistance process for opposing surfactant dissolves, can long storage periods.The atomic layer on polysiloxane surface makes silica nodule have higher morphological stability than conventional liposome, and compared with Nano particles of silicon dioxide, lipid bilayer structure makes it have again lower rigidity and density.Therefore, silica nodule combines the advantage of liposome and silicon dioxide simultaneously, is a kind of desirable pharmaceutical carrier, can meet the demand at present to pharmaceutical carrier.But this stable silica nodule also has following defect: drug release is too slow, valid density can not be maintained at target spot position, oncogenic drug resistance may be led.
Therefore, be necessary to provide a kind of overcome prior art deficiency, combine with high intensity focused ultrasonic beam (HIFU) drug delivery vehicle discharged for controlling hydrophobic drug.
Summary of the invention
Object of the present invention aims to provide one and combines with high intensity focused ultrasonic beam (HIFU) drug delivery vehicle discharged for controlling hydrophobic drug, it has good sensitivity to high intensity focused ultrasonic beam (HIFU) and temperature, and preparation technology is simple, pollution-free, output is high, cost is low and efficiency is high.
Therefore, first aspect, the invention provides a kind of temperature-sensitive silica nodule, and itself and high intensity focused ultrasonic beam (HIFU) combine and discharge for controlling hydrophobic drug, and this temperature-sensitive silica nodule comprises following composition:
Silica nodule forms lipid;
Dipalmitoyl phosphatidyl choline;
MSPC; With
Macrogol 2000-DPPE.
In the temperature-sensitive silica nodule of first aspect, silica nodule forms lipid and has following structure:
In the temperature-sensitive silica nodule of first aspect, the parts by weight of each composition are:
Preferably, the parts by weight of each composition are:
The composition of temperature-sensitive silica nodule of the present invention can also comprise activating agent, and the parts by weight of each composition are:
Wherein, activating agent can be selected from one or more in amycin, paclitaxel, Nile red, Docetaxel, vincristine, cisplatin, lomustine, homoharringtonine, altretamine, aminoglutethimide.
In the temperature-sensitive silica nodule of first aspect, the phase transition temperature of temperature-sensitive silica nodule is 39.0 DEG C-45.0 DEG C.
In the temperature-sensitive silica nodule of first aspect, the vesicle surface covalency of temperature-sensitive silica nodule covers siloxane network skeleton.
In the temperature-sensitive silica nodule of first aspect, the particle size range of temperature-sensitive silica nodule is 50nm-300nm.
Second aspect, the invention provides a kind of method preparing temperature-sensitive silica nodule, the method comprises the steps:
(1) silica nodule is formed lipid to be hydrolyzed in acid ethanol solution, hatch 30min;
(2) chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC and Macrogol 2000-DPPE is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing; Or the chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC, Macrogol 2000-DPPE and activating agent is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing;
(3) solvent in Rotary Evaporators removing step (2) gained mixed liquor is used, dried overnight under vacuo;
(4) water is joined in the solid film that step (3) obtains, carry out aquation in a water bath, then vortex oscillation, finally carry out ultrasonic, temperature-sensitive silica nodule of the present invention can be obtained.
In the method for second aspect, the pH of the acid ethanol solution wherein in step (1) is 3.0, and hatching is carry out at the temperature of 40 DEG C.
In the method for second aspect, the temperature of the water-bath wherein in step (4) is 60 DEG C, and aquation carries out 30min, and vortex oscillation carries out 20min, ultrasonicly carries out 5-10min.
The third aspect, the invention provides temperature-sensitive silica nodule of the present invention and high intensity focused ultrasonic beam (HIFU) be combined in control hydrophobic drug discharge in purposes.
In the purposes of the third aspect, temperature-sensitive silica nodule is applied to patient by following manner:
The spray of intravenous administration, intraarterial delivery, intramuscular administration, Intraperitoneal medication, subcutaneous administration, intra-articular administration, intrathecal drug delivery, intracerebroventricular administration, per nasal, lung suck and oral administration.
In addition, temperature-sensitive silica nodule of the present invention can also be administered to patient by other suitable modes well known to those skilled in the art.
In the purposes of the third aspect, the optimum configurations that use high intensity focused ultrasonic beam (HIFU) is irradiated is: range of voltage values is 185mV-190mV, and duty cycle range is 25%-35%.
In the purposes of the third aspect, the time that high intensity focused ultrasonic beam irradiates the target area of patient is used to be at least 3min.
In the purposes of the third aspect, the target area of patient is irradiated by high intensity focused ultrasonic beam (HIFU) and is heated to 41.0 DEG C-42.0 DEG C.
The present invention, by being incorporated in silica nodule by temperature-sensitive lipid and the lipid with covalently bound peg molecule, has prepared the temperature-sensitive silica nodule combining with high intensity focused ultrasonic beam (HIFU) and discharge for controlling hydrophobic drug.The permeability that this kind of lipid with covalently bound peg molecule can increase film is introduced in expection in silica nodule, discharges fast under HIFU irradiates because the unstability of film and the generation of fenestra can make to contain medicine.The temperature-sensitive silica nodule that bag carries activating agent is effectively delivered into tumor tissues, then irradiates target location by HIFU and produces inertial cavitation and temperature rising, thus cause activating agent to discharge fast from temperature-sensitive silica nodule.
The present invention compared with prior art, at least has following advantage:
(1) the present invention forms lipid and Macrogol 2000-DPPE by adding silica nodule, significantly improves HIFU response performance and the temperature sensitivity of silica nodule.
(2) temperature-sensitive silica nodule of the present invention has higher stability relative to liposome, and the envelop rate of hydrophobic drug is all higher than 80%.
(3) temperature-sensitive silica nodule of the present invention has excellent biocompatibility, and its preparation technology is simple, pollution-free, output is high, cost is low and efficiency is high.The temperature-sensitive silica nodule of gained presents effective HIFU response performance compared to non-HIFU and the siliceous physical ability of responsive to temperature and can significantly improve therapeutic effect, demonstrates wide application prospect.
(4) temperature-sensitive silica nodule of the present invention can ensure that medicine only discharges in the appointed area needing treatment.It can not only increase the local concentration of medicine, reaches maximum therapeutic effect and minimum side effect, but also can avoid the infringement because Formulations for systemic administration causes human body.
(5) medicine that temperature-sensitive silica nodule of the present invention discharges can irradiate with HIFU the heat generation cooperative effect produced.
To sum up, this temperature-sensitive silica nodule system can become by the pharmaceutical carrier of the effective Co ntrolled release behavior of HIFU, will play its important function in hydrophobic drug Co ntrolled release field.
Accompanying drawing explanation
The present invention above-mentioned and/or additional aspect and advantage will become obvious and easy understand from the following description of the accompanying drawings of embodiments, wherein:
Fig. 1 is the transmission electron microscope schematic diagram that bag of the present invention is loaded with the temperature-sensitive silica nodule of paclitaxel;
Fig. 2 is temperature-sensitive silica nodule and the DSPC liposome of the embodiment 1-3 of the variable concentrations adding 0.01-1.0mg/mL in HUVEC cell, hatches the schematic diagram of the cell survival rate after 24 hours;
Fig. 3 is the temperature-sensitive silica nodule of embodiment 1-3 and the half-life test result schematic diagram of DSPC liposome;
Fig. 4 is the variations in temperature schematic diagram that HIFU irradiates the temperature-sensitive silica nodule sample of the lower embodiment of the present invention 1;
Fig. 5 is the release percent schematic diagram over time of temperature-sensitive silica nodule 4 of the present invention paclitaxel under HIFU irradiates;
Fig. 6 is the release percent schematic diagram over time of temperature-sensitive silica nodule 5 of the present invention Nile red under HIFU irradiates;
Fig. 7 is the release percent schematic diagram over time of temperature-sensitive silica nodule 6 of the present invention amycin under HIFU irradiates.
Detailed description of the invention
Be described in further details the present invention below by embodiment, following embodiment is only for illustration of the present invention instead of restriction the present invention.
I. the preparation of temperature-sensitive silica nodule
the preparation of embodiment 1 temperature-sensitive silica nodule 1
Step:
(1) silica nodule is formed lipid to be hydrolyzed in acid ethanol solution, at 40 DEG C, hatch 30min; The pH value of wherein said acid ethanol solution is 3.0; The preparation of acid ethanol solution is by adding in alcoholic solution by dilute hydrochloric acid, and being adjusted to pH value with pH meter is 3.0.
(2) chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC and Macrogol 2000-DPPE is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing;
(3) solvent in Rotary Evaporators removing step (2) gained mixed liquor is used, dried overnight under vacuo;
(4) ultra-pure water of 100ml is joined in the solid film that step (3) obtains, aquation 30min in the water-bath of 60 DEG C; By mixture vortex oscillation 20min, use Probe Ultrasonic Searching 5-10min (30% amplitude exports) subsequently, until obtain finely disseminated solution, i.e. the solution of obtained temperature-sensitive silica nodule 1 of the present invention.
Before measuring the property indices of the solution of temperature-sensitive silica nodule 1, after first gained solution at room temperature being hatched 24 hours, then measure accordingly.
the preparation of embodiment 2 temperature-sensitive silica nodule 2
Preparation method is with embodiment 1, and difference is Probe Ultrasonic Searching 5-10min (20% amplitude exports).
the preparation of embodiment 3 temperature-sensitive silica nodule 3
Preparation method is with embodiment 1, and difference is Probe Ultrasonic Searching 5-10min (10% amplitude exports).
the preparation of embodiment 4 temperature-sensitive silica nodule 4
Step:
(1) silica nodule is formed lipid to be hydrolyzed in acid ethanol solution, at 40 DEG C, hatch 30min; The pH value of wherein said acid ethanol solution is 3.0; The preparation of acid ethanol solution is by adding in alcoholic solution by dilute hydrochloric acid, and being adjusted to pH value with pH meter is 3.0.
(2) chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC, Macrogol 2000-DPPE and paclitaxel is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing;
(3) solvent in Rotary Evaporators removing step (2) gained mixed liquor is used, dried overnight under vacuo;
(4) ultra-pure water of 100ml is joined in the solid film that step (3) obtains, aquation 30min in the water-bath of 60 DEG C; By mixture vortex oscillation 20min, use Probe Ultrasonic Searching 5-10min (20% amplitude exports) subsequently, until obtain finely disseminated solution, i.e. the solution of obtained temperature-sensitive silica nodule 4 of the present invention.
Before measuring the property indices of the solution of temperature-sensitive silica nodule 4, after first gained solution at room temperature being hatched 24 hours, then measure accordingly.
Whole process need carries out under the condition of lucifuge.Paclitaxel centrifugal 10min under the condition of 10,000r/min of unentrapped is separated.It is in the aqueous dispersions of temperature-sensitive silica nodule 4 of 0.2mM that 300 object copper mesh are immersed concentration, after 8min, with filter paper, unnecessary sample is siphoned away, and at room temperature carry out negative staining 5min with the uranyl acetate aqueous solution that the concentration of just preparation is 4% (weight/volume).The copper mesh of absorption temperature-sensitive silica nodule 4 uses distilled water wash subsequently, and dries in atmosphere.Finally, adopt transmission electron microscope (model H-7650) at the transmission electron microscope schematic diagram of the accelerating potential acquisition sample of 100 kilovolts.Transmission electron microscope schematic diagram clearly confirms the formation of spherical vesicles, and result as shown in Figure 1.
the preparation of embodiment 5 temperature-sensitive silica nodule 5
Preparation method is with embodiment 4.
the preparation of embodiment 6 temperature-sensitive silica nodule 6
Preparation method is with embodiment 4.
the preparation of embodiment 7 temperature-sensitive silica nodule 7
Preparation method is with embodiment 4.
the preparation of embodiment 8 temperature-sensitive silica nodule 8
Preparation method is with embodiment 4.
the preparation of embodiment 9 temperature-sensitive silica nodule 9
Preparation method is with embodiment 4.
the preparation of embodiment 10 temperature-sensitive silica nodule 10
Preparation method is with embodiment 4.
the preparation of embodiment 11 temperature-sensitive silica nodule 11
Preparation method is with embodiment 4.
the preparation of embodiment 12 temperature-sensitive silica nodule 12
Preparation method is with embodiment 4.
the preparation of embodiment 13 temperature-sensitive silica nodule 13
Preparation method is with embodiment 4.
II. the performance test of temperature-sensitive silica nodule
embodiment 14 phase transition temperature is tested
Temperature-sensitive silica nodule phase transition temperature (T
m) mensuration of value completed by differential scanning calorimetry (DSC).DSC is one of the most widely used method of one, for studying the thermal behavior of bilayer lipid system.The Excess heat capacity of duplicature to the function of temperature at gel-liquid-crystal phase-transition temperature T
mthe enthalpy change of maximum is revealed with the table of integrals of the area under easement curve.
The concrete operation step measured is as follows: before sample is measured, appliance requires first intensification preheating is about 1h; Secondly, respectively the temperature-sensitive silica nodule sample of the embodiment 1-13 of 10-15 μ L and suitable reference solution (reference solution: deionized water to) are joined in an aluminum pot, use tweezers to be placed on by aluminium lid on pot and seal.Pot dish is put into instrument, applies outside nitrogen current simultaneously.With the tachometric survey T of intensification per minute 0.5 DEG C in the interested temperature range of specifying
mvalue.When maximum hot-fluid reaches the programming rate of predetermined 0.5 DEG C per minute, the temperature of namely getting corresponding to this point is the phase transition temperature of respective sample.The results are shown in Table 1.
The phase transition temperature test result of the temperature-sensitive silica nodule sample of table 1 embodiment 1-13
| Numbering | Sample | Phase transition temperature |
| 1 | Embodiment 1 | 40.7±1.1 |
| 2 | Embodiment 2 | 43.1±1.7 |
| 3 | Embodiment 3 | 42.2±1.5 |
| 4 | Embodiment 4 | 41.4±2.0 |
| 5 | Embodiment 5 | 40.2±0.7 |
| 6 | Embodiment 6 | 44.1±1.1 |
| 7 | Embodiment 7 | 45.0±0.8 |
| 8 | Embodiment 8 | 43.3±0.5 |
| 9 | Embodiment 9 | 41.2±1.5 |
| 10 | Embodiment 10 | 42.5±2.4 |
| 11 | Embodiment 11 | 43.1±1.2 |
| 12 | Embodiment 12 | 40.5±1.3 |
| 13 | Embodiment 13 | 39.0±0.5 |
embodiment 15 particle diameter is tested
Compound concentration is the aqueous dispersions of the temperature-sensitive silica nodule of the embodiment 1-13 of 0.1mM respectively, is measured particle diameter and the current potential size of each sample by 90Plus/BI-MAS granularity potentiometer (Brooker Hai Wen instrument company, the U.S.).The results are shown in Table 2.
Table 2 particle diameter test result
| Numbering | Sample | Particle diameter (nm) | Zeta potential (mV) |
| 1 | Embodiment 1 | 50.00±2.1 | -25.5±3.8 |
| 2 | Embodiment 2 | 195.3±10.7 | -26.1±3.1 |
| 3 | Embodiment 3 | 300.0±12.5 | -31.8±4.1 |
| 4 | Embodiment 4 | 196.4±3.7 | -25.9±2.4 |
| 5 | Embodiment 5 | 190.6±8.7 | -28.2±3.5 |
| 6 | Embodiment 6 | 188.9±7.3 | -29.8± 3.4 |
| 7 | Embodiment 7 | 175.5±9.8 | -32.1±3.1 |
| 8 | Embodiment 8 | 185.3±7.5 | -28.5±5.7 |
| 9 | Embodiment 9 | 176.8±5.5 | -30.8±2.9 |
| 10 | Embodiment 10 | 182.7±8.4 | -31.4±2.5 |
| 11 | Embodiment 11 | 195.2±7.2 | -30.5±5.5 |
| 12 | Embodiment 12 | 176.5±6.3 | -25.7±5.3 |
| 13 | Embodiment 13 | 190.3±8.5 | -28.5±4.2 |
embodiment 16 stability experiment
Measure the stability of temperature-sensitive silica nodule corresponding to specific embodiment 1-13 respectively.
By adding the surfactant TritonX-100 (TX-100) of different proportion in temperature-sensitive silica nodule solution corresponding to embodiment 1-13, measure the change of particle diameter, and change with the traditional liposomal particle diameter under equal conditions (nm) that DSPC makes compare the stability investigating temperature-sensitive silica nodule, result is as shown in table 3.
Wherein the preparation method of DSPC liposome is as follows:
126mg distearoyl phosphatidylcholine (DSPC) phospholipid is dissolved in chloroform, uses Rotary Evaporators to steam and desolventize, form uniform thin film at flask inwall, and dried overnight under vacuo; The ultra-pure water of 100ml is joined in above-mentioned obtained solid film, aquation 30min in the water-bath of 60 DEG C; By mixture vortex oscillation 20min, use Probe Ultrasonic Searching 5-10min (20% amplitude exports) subsequently, until obtain finely disseminated solution, i.e. obtained DSPC liposome solutions.
The stability experiment result of the temperature-sensitive silica nodule that table 3 embodiment 1-13 is corresponding
As can be seen from the above table, when after the TX-100 aqueous solution being added to 25 times amount, the size of temperature-sensitive silica nodule remains unchanged substantially, and traditional liposomal prepared by DSPC is after adding the TX-100 aqueous solution of 5 times amount, its particle diameter obviously reduces, illustrate that its imitated vesicle structure is destroyed, thus prove that temperature-sensitive silica nodule of the present invention has stability more better than traditional liposomal.
embodiment 17 envelop rate and drug loading measure
By prepared medicine carrying temperature-sensitive silica nodule, namely the temperature-sensitive silica nodule sample of embodiment 4-13 is centrifugal, gets supernatant, by the content of each activating agent of high effective liquid chromatography for measuring.
The every condition determination selected in actual experiment is as follows: C-18 reversed phase chromatographic column: 150 × 4.6mm, sample concentration 5 μMs; Sample size: 20 μ L; Mobile phase: first alcohol and water (by volume 7:3); Mobile phase speed: 1.0mL/min.Following formulae discovery is utilized to obtain envelop rate and the drug loading of the temperature-sensitive silica nodule of embodiment 4-13 afterwards.
The results are shown in Table 4.
The envelop rate of the temperature-sensitive silica nodule of table 4 embodiment 4-13 and drug loading result
| Numbering | Sample | Envelop rate % | Drug loading % |
| 1 | Embodiment 4 | 81.3±6.8％ | 3.4±0.5％ |
| 2 | Embodiment 5 | 83.2±5.7％ | 3.5±0.4％ |
| 3 | Embodiment 6 | 88.5±4.3％ | 3.6±0.7％ |
| 4 | Embodiment 7 | 84.3±3.8％ | 3.4±0.6％ |
| 5 | Embodiment 8 | 80.5±5.5％ | 3.1±0.3％ |
| 6 | Embodiment 9 | 78.6±3.1％ | 2.9±0.4％ |
| 7 | Embodiment 10 | 82.6±4.5％ | 3.4±0.21％ |
| 8 | Embodiment 11 | 82.7±3.5％ | 3.5±0.52％ |
| 9 | Embodiment 12 | 85.6±2.8％ | 3.5±0.21％ |
| 10 | Embodiment 13 | 83.7±6.5％ | 3.3±0.72％ |
embodiment 18 biocompatibility measures
By 1 × 10
4individual HUVEC cells/well is inoculated in 96 orifice plates.Spend the night after increasing, cell is used for experiment.The temperature-sensitive silica nodule sample culture medium of embodiment 1-3 is diluted to certain sample concentration, and adds in the hole of specifying, make final sample concentration be 0.01,0.05,0.1,0.2,0.5 and 1.0mg/mL.Then, in dark, the above-mentioned cell adding sample is positioned in cell culture incubator and hatches 24h, rinse various kinds sample wells three times with freshly prepd aseptic PBS afterwards.Being dissolved with aseptic PBS by MTT makes its concentration be 5mg/mL, adds 20 μ L toward each hole in 96 orifice plates, and further at 37 DEG C, 5%CO
2incubator in hatch 4h.Afterwards, remove the culture medium in each hole carefully and add dimethyl sulfoxide 150 μ L.Microplate reader is used to measure the absorbance at 490nm place.The survival rate representing cell with blood serum medium cultured cells is 100%.Each concentration has 5 Duplicate Samples, and each experiment repeats 3 times.Experimental result as shown in Figure 2.As can be seen from Figure 2, when the concentration of temperature-sensitive silica nodule reaches 1.0mg/mL the survival rate of HUVEC cell still more than 80%.
Investigate the biocompatibility of DSPC liposome under the same conditions.Result shows that the biocompatibility of temperature-sensitive silica nodule of the present invention is similar to DSPC liposome.
the mensuration of embodiment 19 half-life
Extracting male Wistar rat 3, tail vein injection saline, after 5min, heart extracting blood, is placed in the centrifuge tube of heparin process, and-20 DEG C of stored frozen are for subsequent use.Extracting male Wistar rat 3, implement the operation of jugular vein intubate, fasting, freely drink water after 12h, tail vein injects the dispersion liquid of the temperature-sensitive silica nodule of embodiment 1-3 and the dispersion liquid (concentration: 20mg/mL, injected dose: 0.5mL/ only) of DSPC liposome respectively.
Respectively at after administration 2,3,4,6,12,24h gets blood, the blood sample volume of each time point collection is 0.3ml, is placed in the centrifuge tube of heparin process, and-20 DEG C of stored frozen are for subsequent use.Often blood sampling point blood sample (300 μ L), is first mixed and heated to liquid with 1mL perchloric acid all dry.And then mix with 1mL concentrated nitric acid, be settled to 5mL heat 1h at 70 DEG C after as liquid to be measured.In sample, silicon content measures through inductivity coupled plasma mass spectrometry (ICP-MS), and experimental result as shown in Figure 3.As seen from the figure, the temperature-sensitive silica nodule of embodiment of the present invention 1-3 compares DSPC liposome, and its circulation time in vivo significantly increases.
embodiment 20HIFU irradiates the change of lower temperature
Utilize HIFU to irradiate the solution of the temperature-sensitive silica nodule sample of the embodiment of the present invention 1, initial temperature is set as 37 DEG C.Instrument parameter: voltage is 185-190mV, dutycycle (DC) is 25-35%, HIFU irradiation different time, and the result of variations of temperature as shown in Figure 4.
As we can see from the figure, when voltage is 185mV, when irradiation time reaches 3 minutes, its temperature rises to 41.3 DEG C, when voltage is 190mV, irradiate 3 minutes, its temperature rises to 41.8 DEG C, continues to extend irradiation time, and sample solution temperature maintains 42 DEG C substantially, therefore, the irradiation time of HIFU at least needs 3min.
III. temperature-sensitive silica nodule of the present invention is combined with HIFU, for controlling the release of medicine
Utilize HIFU to irradiate the sample solution of the temperature-sensitive silica nodule of embodiment 4-6, initial temperature is set as 37 DEG C.Instrument parameter: voltage is 185-190mV, dutycycle (DC) is 25-35%, HIFU irradiation different time, the drug release percent of test each sample.Result as illustrated in figs. 5-7.
Can be clear that from Fig. 5-7, when not carrying out (now the temperature of sample is 37 DEG C) when HIFU irradiates to sample, slowly, in 10min, the release percent of medicine is all below 5% for the rate of release of medicine; And when carrying out HIFU to sample and irradiating (now the temperature of sample is 42 DEG C), the rate of release of medicine improves greatly, in 10min, the release percent of medicine is between 20-30%, and this result absolutely proves that temperature-sensitive silica nodule of the present invention has good HIFU response performance.
Above certain preferred embodiments of the present invention is described.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (17)
1. a temperature-sensitive silica nodule, it is combined with high intensity focused ultrasonic beam and discharges for controlling hydrophobic drug, and described temperature-sensitive silica nodule comprises following composition:
Silica nodule forms lipid;
Dipalmitoyl phosphatidyl choline;
MSPC; With
Macrogol 2000-DPPE.
2. temperature-sensitive silica nodule according to claim 1, wherein said silica nodule forms lipid and has following structure:
3. temperature-sensitive silica nodule according to claim 1 and 2, the parts by weight of wherein said each composition are:
4. temperature-sensitive silica nodule according to claim 3, the parts by weight of wherein said each composition are:
5. temperature-sensitive silica nodule according to claim 3, the composition of wherein said temperature-sensitive silica nodule also comprises activating agent, and the parts by weight of each composition are:
6. temperature-sensitive silica nodule according to claim 5, wherein said activating agent be selected from amycin, paclitaxel, Nile red, Docetaxel, vincristine, cisplatin, lomustine, homoharringtonine, altretamine, aminoglutethimide one or more.
7. temperature-sensitive silica nodule according to claim 1, the phase transition temperature of wherein said temperature-sensitive silica nodule is 39.0 DEG C-45.0 DEG C.
8. temperature-sensitive silica nodule according to claim 1, the vesicle surface covalency of wherein said temperature-sensitive silica nodule covers siloxane network skeleton.
9. temperature-sensitive silica nodule according to claim 1, the particle size range of wherein said temperature-sensitive silica nodule is 50nm-300nm.
10. prepare a method for the temperature-sensitive silica nodule according to any one of claim 1 to 9, described method comprises the steps:
(1) silica nodule is formed lipid to be hydrolyzed in acid ethanol solution, hatch 30min;
(2) chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC and Macrogol 2000-DPPE is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing; Or the chloroformic solution being dissolved with dipalmitoyl phosphatidyl choline, MSPC, Macrogol 2000-DPPE and activating agent is joined in the hydrolyzed solution of step (1), Homogeneous phase mixing;
(3) solvent in Rotary Evaporators removing step (2) gained mixed liquor is used, dried overnight under vacuo;
(4) water is joined in the solid film that step (3) obtains, carry out aquation in a water bath, then vortex oscillation, finally carry out ultrasonic, described temperature-sensitive silica nodule can be obtained.
11. methods according to claim 10, the pH of the described acid ethanol solution wherein in step (1) is 3.0, described in hatch be carry out at the temperature of 40 DEG C.
12. methods according to claim 10, the temperature of the described water-bath wherein in step (4) is 60 DEG C, and described aquation carries out 30min, and described vortex oscillation carries out 20min, describedly ultrasonicly carries out 5-10min.
13. temperature-sensitive silica nodules according to any one of claim 1 to 9 and high intensity focused ultrasonic beam be combined in control hydrophobic drug discharge in purposes.
14. purposes according to claim 13, wherein said temperature-sensitive silica nodule is applied to patient by following manner:
The spray of intravenous administration, intraarterial delivery, intramuscular administration, Intraperitoneal medication, subcutaneous administration, intra-articular administration, intrathecal drug delivery, intracerebroventricular administration, per nasal, lung suck and oral administration.
15. purposes according to claim 13 or 14, the optimum configurations wherein using described high intensity focused ultrasonic beam to irradiate is: range of voltage values is 185mV-190mV, and duty cycle range is 25%-35%.
16. purposes according to claim 15, wherein use the time that described high intensity focused ultrasonic beam irradiates the target area of patient to be at least 3min.
17. purposes according to claim 16, the target area of wherein said patient is irradiated by described high intensity focused ultrasonic beam and is heated to 41.0 DEG C-42.0 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112546062A (en) * | 2020-10-27 | 2021-03-26 | 中国科学院生物物理研究所 | Perfluorocarbon silicon plastid and preparation method and application thereof |
| CN115093858A (en) * | 2022-07-01 | 2022-09-23 | 中国矿业大学 | Temperature-sensitive silica body inhibitor for preventing coal spontaneous combustion and preparation method and application thereof |
| CN116603101A (en) * | 2023-07-20 | 2023-08-18 | 北京大学第三医院（北京大学第三临床医学院） | Gold-siliceous body-polycaprolactone scaffold system and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112546062A (en) * | 2020-10-27 | 2021-03-26 | 中国科学院生物物理研究所 | Perfluorocarbon silicon plastid and preparation method and application thereof |
| CN112546062B (en) * | 2020-10-27 | 2022-08-19 | 中国科学院生物物理研究所 | Perfluorocarbon silicon plastid and preparation method and application thereof |
| CN115093858A (en) * | 2022-07-01 | 2022-09-23 | 中国矿业大学 | Temperature-sensitive silica body inhibitor for preventing coal spontaneous combustion and preparation method and application thereof |
| CN116603101A (en) * | 2023-07-20 | 2023-08-18 | 北京大学第三医院（北京大学第三临床医学院） | Gold-siliceous body-polycaprolactone scaffold system and preparation method and application thereof |
| CN116603101B (en) * | 2023-07-20 | 2023-10-03 | 北京大学第三医院（北京大学第三临床医学院） | Gold-siliceous body-polycaprolactone scaffold system and preparation method and application thereof |
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